Abstract

LB-83

We optimized this assay by using the Xenogen IVIS imaging system to be more efficient and a high throughput in vivo screening model. Here, we explored what was an optimal cell density in hollow fibers. PVDF hollow fibers (500,000 Da molecular weight cut-off, 1 .O mm ID), a human melanoma cell line A375-luc and a test compound were used. Four cell densities, 1,000, 3,000, 9,000 and 27,000 cells in a 2-cm length PVDF fiber (a volume of 20 µl) were utilized in the experiment. Net growth and treatment response of tumor cells in the fibers was analyzed on days 1, 2, 3, and 4 post-treatment in CD-1 nude mice. In general, after hollow fibers filled with the cells were incubated at 37º C for 24 hours, the study fibers were subcutaneously (SC fibers) and intraperitoneally (IP fibers) implanted into animals. The study animals were orally dosed by either the test compound 50 mg/kg or vehicle twice daily for four days. 6 animals, including 3 from vehicle and 3 from treated, were sacrificed on day 2, 3, 4 and 5 respectively; the fibers were removed from animals and immediately put into a culture medium containing luciferin 150 mg/ml for 30 minutes and ex-vivo imaging was conducted. The bioluminescent imaging data were analyzed by using Xenogen IVIS system and the net growth of the tumor cells was calculated. The compound efficacy was assessed by % T/C (T: treated and C: control). The study found that the tumor cells in the IP fibers exhibited distinct growth feature among the density groups: exponential/1,000 cells on day 1-4 (% net growth: 142%/day 2, 509%/day 3 and 882%/day 4); exponential/3,000 cells on day 1-3 and flat on day 4 (% net growth: 143%/day 2, 538%/day 3 and 544%/day 4); flat/9,000 cells on days 1-4 (% net growth: 163%/day 2, 186%/day 3 and 149%/ day 4); irregular/27,000 cells on day 1-4 (% net growth: -7.5%/day 2, 57%/day 3 and 54%/day 4). The tumor cells in the SC fibers showed a similar growth pattern as the IP fibers, but sluggishly as follows: 1,000 cells (% net growth: 60%/day 2, 268%/day 3 and 672%/day 4); 3,000 cells (% net growth: 99%/day 2, 317%/day 3 and 308%/day 4); 9,000 cells (% net growth: 64%/day 2, 61%/day 3 and 123%/day 4); 27,000 cells (% net growth: 20%/day 2, 5.3%/day 3 and 65.3%/day 4). We also found that the maximum inhibition of the test compound on the A375 tumor cells was 11-14% (%T/C) and the tumor cells displayed particular times linked with cell densities to reach the maximum inhibition: day 4/1,000 cells, day 3/3,000 cells, day 2/9,000 cells and day 1/27,000 cells (%T/C from IP fibers: 14%/1,000 cells/day 4, 11%/3,000 cells/day 3, 14%/9,000 cells/day 2 and 33%/27,000 cells/day 1; %T/C from SC fibers: 11%/1,000 cell/day 4, 18%/3,000 cells/day 3, 33%/9,000 cells/day 2 and 34%/27,000 cells/day 1). But here we need to emphasize that the highest density group of 27,000 cells was too high to get reliable data. Our findings provided evidences that optimal cell density played a critical role associated with adequate study length in the hollow fiber assay.